Genetic susceptibility in tuberculosis.

Yim JJ, Selvaraj P.

Division of Pulmonary and Critical Care Medicine, Department of Internal Medicine

and Lung Institute, Seoul National University College of Medicine, Seoul,

Republic of Korea.

The importance of host genetic factors in determining susceptibility to

tuberculosis (TB) has been studied extensively using various methods, such as

case-control, candidate gene and genome-wide linkage studies. Several important

candidate genes like human leucocyte antigen/alleles and non-human leucocyte

antigen genes, such as cytokines and their receptors, chemokines and their

receptors, pattern recognition receptors (including toll-like receptors, mannose

binding lectin and the dendritic cell-specific intercellular adhesion molecule-3

grabbing nonintegrin), solute carrier family 11A member 1 (formerly known as

natural resistance-associated macrophage protein 1) and purinergic P2X7 receptor

gene polymorphisms, have been associated with differential susceptibility to TB

in various ethnic populations. This heterogeneity has been explained by

host-pathogen and gene-environment interactions and evolutionary selection

pressures. Although the achievements of genetics studies might not yet have

advanced the prevention and treatment of TB, researchers have begun to widen

their scope of investigation to encompass these practical considerations.

PMID: 20199642 [PubMed - in process]

 

Advances in the diagnosis of tuberculosis.

Lange C, Mori T.

Clinical Infectious Diseases, Research Center Borstel, Borstel, Germany.

Tuberculosis ranges among the leading causes of morbidity and mortality

worldwide. A diagnostic approach to a patient with possible tuberculosis includes

a detailed medical history and clinical examination as well as radiological,

microbiological, immunological, molecular-biological and histological

investigations, where available. Recently, important advances have been achieved

in these fields that have led to substantial improvements in the accuracy and the

timing of the diagnosis of tuberculosis. Novel methods allow for a better

identification of latently infected individuals who are at risk of developing

active tuberculosis, they also offer the possibility for a rapid diagnosis of

active tuberculosis in patients with negative sputum smears for acid-fast bacilli

and enable prompt identification of drug-resistant strains of Mycobacterium

tuberculosis directly from respiratory specimen with a high accuracy. In

addition, promising methods that will further optimize the diagnosis of

tuberculosis are under development. In the future, therapeutic interventions

based on the results of novel diagnostic procedures can be made earlier leading

to improvements in patient care.

PMID: 20199641 [PubMed - in process]

 

Identification of HLA-DR4-restricted T-cell epitope on MPT51 protein, a major

secreted protein derived from Mycobacterium tuberculosis using MPT51 overlapping

peptides screening and DNA vaccination.

Wang LX, Nagata T, Tsujimura K, Uchijima M, Seto S, Koide Y.

Department of Infectious Diseases, Hamamatsu University School of Medicine,

1-20-1 Higashi-ku, Handa-yama, Hamamatsu 431-3192, Japan.

We identified a novel HLA-DR4-restricted CD4+ T-cell epitope on a secreted

antigen of Mycobacterium tuberculosis, MPT51, in 004149-MM HLA-DR4-transgenic

mice which express HLA-DRB1*0401, but not murine MHC class II molecules. The mice

were immunized with plasmid DNA encoding MPT51 using gene gun and interferon

(IFN)-gamma production from the immune splenocytes was analyzed. In response to

overlapping synthetic peptides covering the mature MPT51 sequence, only one

peptide, p191-210, stimulated the splenocytes to produce IFN-gamma. Further

analysis using flow cytometry and computer-assisted algorithm, ProPred, narrowed

down the region of CD4+ T-cell epitope to p191-202. The CD4+ T-cell epitope would

be feasible for vaccine design against tuberculosis as well as for analysis of

MPT51-specific T-cells in M. tuberculosis infection. Copyright 2009 Elsevier Ltd.

All rights reserved.

PMID: 20188259 [PubMed - in process]

 

Timing of initiation of antiretroviral drugs during tuberculosis therapy.

Abdool Karim SS, Naidoo K, Grobler A, Padayatchi N, Baxter C, Gray A, Gengiah T,

Centre for the AIDS Programme of Research in South Africa, University of

KwaZulu-Natal, Durban, South Africa. caprisa@ukzn.ac.za

BACKGROUND: The rates of death are high among patients with coinfection with

tuberculosis and the human immunodeficiency virus (HIV). The optimal timing for

the initiation of antiretroviral therapy in relation to tuberculosis therapy

remains controversial. METHODS: In an open-label, randomized, controlled trial in

Durban, South Africa, we assigned 642 patients with both tuberculosis and HIV

infection to start antiretroviral therapy either during tuberculosis therapy (in

two integrated-therapy groups) or after the completion of such treatment (in one

sequential-therapy group). The diagnosis of tuberculosis was based on a positive

sputum smear for acid-fast bacilli. Only patients with HIV infection and a CD4+

cell count of less than 500 per cubic millimeter were included. All patients

received standard tuberculosis therapy, prophylaxis with

trimethoprim-sulfamethoxazole, and a once-daily antiretroviral regimen of

didanosine, lamivudine, and efavirenz. The primary end point was death from any

cause. RESULTS: This analysis compares data from the sequential-therapy group and

the combined integrated-therapy groups up to September 1, 2008, when the data and

safety monitoring committee recommended that all patients receive integrated

antiretroviral therapy. There was a reduction in the rate of death among the 429

patients in the combined integrated-therapy groups (5.4 deaths per 100

person-years, or 25 deaths), as compared with the 213 patients in the

sequential-therapy group (12.1 per 100 person-years, or 27 deaths); a relative

reduction of 56% (hazard ratio in the combined integrated-therapy groups, 0.44;

95% confidence interval, 0.25 to 0.79; P=0.003). Mortality was lower in the

combined integrated-therapy groups in all CD4+ count strata. Rates of adverse

events during follow-up were similar in the two study groups. CONCLUSIONS: The

initiation of antiretroviral therapy during tuberculosis therapy significantly

improved survival and provides further impetus for the integration of

tuberculosis and HIV services. (ClinicalTrials.gov number, NCT00398996.) 2010

Massachusetts Medical Society

PMID: 20181971 [PubMed - indexed for MEDLINE]

 

Linezolid use for treatment of multidrug-resistant and extensively drug-resistant

tuberculosis, New York City, 2000-06.

Anger HA, Dworkin F, Sharma S, Munsiff SS, Nilsen DM, Ahuja SD.

New York City Department of Health and Mental Hygiene, Bureau of Tuberculosis

Control, 225 Broadway, 22nd floor, New York, NY 10007, USA.

Rationale Linezolid may be effective for the treatment of multidrug-resistant

(MDR) and extensively drug-resistant (XDR) tuberculosis (TB); however, serious

adverse events are common and there is little information on the management of

these toxicities. Methods We retrospectively reviewed public health and medical

records of 16 MDR TB patients, including 10 patients with XDR TB, who were

treated with linezolid in New York City between January 2000 and December 2006,

to determine treatment outcomes and describe the incidence, management and

predictors of adverse events. Results Linezolid was added to MDR TB regimens for

a median duration of 16 months (range: 1-29). Eleven patients (69%) completed

treatment, four (25%) died and one (6%) discontinued treatment without relapse.

Myelosuppression occurred in 13 (81%) patients a median of 5 weeks (range: 1-11)

after starting linezolid, gastrointestinal adverse events occurred in 13 (81%)

patients after a median of 8 weeks (range: 1-57) and neurotoxicity occurred in

seven (44%) patients after a median of 16 weeks (range: 10-111). Adverse events

were managed by combinations of temporary suspension of linezolid, linezolid dose

reduction and symptom management. Five (31%) patients required eventual

discontinuation of linezolid. Myelosuppression was more responsive to clinical

management strategies than was neurotoxicity. Leucopenia and neuropathy occurred

more often in males and older age was associated with thrombocytopenia (P <

0.05). Conclusions The majority of MDR TB patients on linezolid had favourable

treatment outcomes, although treatment was complicated by adverse events that

required extensive clinical management.

PMID: 20150181 [PubMed - in process]

 

MVA-85A, a novel candidate booster vaccine for the prevention of tuberculosis in

children and adults.

Nicol MP, Grobler LA.

University of Cape Town, Institute of Infectious Diseases and Molecular Medicine,

South Africa. Mark.Nicol@uct.ac.za

MVA-85A, in development by Oxford-Emergent Tuberculosis Consortium Ltd and the

EU-funded research program TB-VAC, is a live attenuated viral vaccine expressing

the immunodominant tuberculosis (TB) antigen 85A, and is intended for use in a

heterologous prime-boost strategy to prevent TB. MVA-85A is highly immunogenic in

both animals and humans, eliciting strong polyfunctional CD4+ T-cell responses

when administered as a boost following BCG vaccination or when administered to

individuals previously exposed to TB. Animal studies have demonstrated trends

toward reduced pathology and bacillary burden for animals vaccinated with BCG

prime followed by MVA-85A boost compared with BCG alone; however, these positive

effects appear to be modest, and interpretation is limited by the small number of

animals tested. The vaccine has an excellent safety profile in BCG-naïve,

previously BCG-vaccinated and TB-exposed adults, as well as in BCG-vaccinated

adolescents and children. At the time of publication, MVA-85A was in a more

advanced stage of clinical development than other novel TB vaccine candidates,

with a large-scale, proof-of-concept phase IIb clinical trial underway for the

determination of safety, immunogenicity and prevention of TB in infants.

PMID: 20140824 [PubMed - in process]

 

Evaluating the non-tuberculous mycobacteria effect in the tuberculosis infection

diagnosis.

Latorre I, De Souza-Galvão M, Ruiz-Manzano J, Lacoma A, Prat C, Altet N,

Servei de Microbiologia, Fundació Institut d'Investigació en Ciències de la Salut

Germans Trias i Pujol, Carretera del Canyet s/n, 08916 Badalona Barcelona Spain.

The aim of the present study was to determine the role of previous

non-tuberculous mycobacteria sensitisation in children as a factor of discordant

results between tuberculin skin test (TST) and an in vitro T-cell based assay

(T-SPOT.TB; Oxford Immunotec, Oxford, UK). We enrolled 21 non-bacille

Calmette-Guérin-vaccinated paediatric patients for suspicious of latent

tuberculosis infection (LTBI). These patients yielded a positive TST and a

negative T-SPOT.TB. Cells were stimulated with Mycobacterium avium sensitin

(having cross-reaction with Mycobacterium intracellulare and Mycobacterium

scrofulaceum) and the presence of reactive T-cells was determined by an ex vivo

ELISPOT. From the 21 patients, in 10 cases (47.6%), we obtained a positive

ELISPOT result after stimulation with M. avium sensitin, in six (28.6%) cases,

the result was negative and in the remaining five (23.8%) cases, the result was

indeterminate. In conclusion, previous non-tuberculous mycobacteria sensitisation

induces false-positive results in the TST for diagnosing LTBI and the use of

gamma-interferon tests could avoid unnecessary chemoprophylaxis treatment among a

child population.

PMID: 20123845 [PubMed - in process]

 

Associations between Mycobacterium tuberculosis strains and phenotypes.

Brown T, Nikolayevskyy V, Velji P, Drobniewski F.

United Kingdom Health Protection Agency, London UK.

To inform development of tuberculosis (TB) control strategies, we characterized a

total of 2,261 Mycobacterium tuberculosis complex isolates by using multiple

phenotypic and molecular markers, including polymorphisms in repetitive sequences

(spoligotyping and variable-number tandem repeats [VNTRs]) and large sequence and

single-nucleotide polymorphisms. The Beijing family was strongly associated with

multidrug resistance (p = 0.0001), and VNTR allelic variants showed strong

associations with spoligotyping families: >or=5 copies at exact tandem repeat

(ETR) A, >or=2 at mycobacterial interspersed repetitive unit 24, and >or=3 at

ETR-B associated with the East African-Indian and M. bovis strains. All M.

tuberculosis isolates were differentiated into 4 major lineages, and a maximum

parsimony tree was constructed suggesting a more complex phylogeny for M.

africanum. These findings can be used as a model of pathogen global diversity.

PMID: 20113558 [PubMed - in process]

 

Nosocomial transmission of the F15/LAM4/KZN genotype of Mycobacterium

tuberculosis in patients on tuberculosis treatment.

Pillay M, Sturm AW.

Department of Medical Microbiology, Nelson R Mandela School of Medicine,

University of KwaZulu-Natal, Durban, South Africa.

SETTING: King George V (KGV) Hospital has the largest tuberculosis (TB) facility

in KwaZulu-Natal (KZN), the province with the highest prevalence of TB-HIV (human

immunodeficiency virus) co-infection in South Africa. During the study, KGV was

the only provincial referral hospital for patients with drug-resistant TB.

OBJECTIVE: To determine the role of nosocomial transmission in patients infected

with a new strain of Mycobacterium tuberculosis during treatment. DESIGN:

Insertion sequence 6110-DNA fingerprinting was performed on stored isolates from

patients with culture-positive pulmonary TB for more than 6 weeks after treatment

started and those who relapsed. RESULTS AND CONCLUSION: DNA fingerprints of 14 of

26 patients with differing isolates matched those of other patients. Four of them

acquired a F15/LAM4/KZN genotype, while two acquired fully susceptible Beijing

strains. Three of the four F15/LAM4/KZN strains were multidrug-resistant with

identical fingerprint patterns, while the fourth was fully susceptible. One of

these was acquired during hospitalisation and three after discharge. Both

HIV-infected and non-infected patients are at risk of infection with the

F15/LAM4/KZN strain in health care facilities and within the community. Rapid

diagnostic tests, separation of TB and non-TB patients on admission and isolation

of multidrug-resistant and extensively drug-resistant TB patients are essential

to curb nosocomial transmission.

PMID: 20074415 [PubMed - in process]

 

Fluoroquinolone resistance in renal isolates of Mycobacterium tuberculosis.

Webster D, Long R, Shandro C, Pettipas J, Leblanc J, Davidson R, Fanning A.

Department of Internal Medicine, Saint John Regional Hospital, Saint John, New

Brunswick, Canada. dvwebste@dal.ca

SETTING: Alberta, Canada, 1990-2003. OBJECTIVE: Monotherapy of active

tuberculosis (TB) promotes drug resistance. Given the common practice of empiric

fluoroquinolone (FQ) therapy for urinary tract infections (UTI) and frequent

delayed diagnosis of renal TB, we assessed urine Mycobacterium tuberculosis

isolates for FQ resistance. DESIGN: Retrospective study. Urine M. tuberculosis

isolates underwent FQ susceptibility testing. Records were reviewed for evidence

of FQ exposure and diagnostic delay. RESULTS: Among 78 culture-positive renal TB

patients between 1990 and 2003, initial isolates of M. tuberculosis were

available from 74 (94.9%). Three (4.1%) were FQ-resistant. Previous FQ use was

confirmed in nine cases (12.2%). FQ-exposed isolates were more likely than

non-exposed isolates to be FQ-resistant (2/9, 22.2% vs. 1/65, 1.5%, P = 0.037).

Among 41 cases (55.4%) with signs or symptoms of UTI, eight (19.5%) had previous

FQ exposure, of which seven (87.5%) had delayed diagnosis. Only 15/33 (45.5%) UTI

symptomatic cases without prior FQ exposure had delayed diagnosis (P = 0.050). In

2/8 (25%) UTI symptomatic cases with prior FQ exposure, the M. tuberculosis

isolate was FQ-resistant. CONCLUSION: FQ monotherapy of unsuspected renal TB may

delay diagnosis and lead to FQ resistance.

PMID: 20074414 [PubMed - in process]

 

Neuroradiological features of the tuberculosis-associated immune reconstitution

inflammatory syndrome.

Marais S, Scholtz P, Pepper DJ, Meintjes G, Wilkinson RJ, Candy S.

Department of Medicine, GF Jooste Hospital, Manenberg, South Africa.

suzaanmarais@gmail.com

SETTING: Paradoxical tuberculosis-associated immune reconstitution inflammatory

syndrome (TB-IRIS) is an important complication in human immunodeficiency virus

type I (HIV-1) infected tuberculosis (TB) patients who start combination

antiretroviral treatment (ART). Neurological manifestations occur in more than

10% of TB-IRIS cases. Apart from a few case reports, the radiological features of

neurological TB-IRIS have not been described. OBJECTIVE: To describe the

neuroradiological findings of patients with paradoxical neurological TB-IRIS.

DESIGN: Computed tomography (CT; n = 13) and magnetic resonance imaging (n = 3)

findings of 16 patients were reviewed. RESULTS: IRIS manifestations included

meningitis (n = 4), intracranial space occupying lesions (SOLs, presumed

tuberculomas; n = 5), meningitis and SOLs (n = 5), radiculomyelitis (n = 1) and

spondylitis (n = 1). In patients with tuberculoma IRIS, we observed a high

prevalence of 1) low density lesions on non-contrast-enhanced CT (all lesions),

2) multiple lesions (in 5/10 patients) and 3) perilesional oedema (17/22

lesions). In patients with meningitis, meningeal enhancement (n = 2) and

hydrocephalus (n = 1) were infrequently observed. CONCLUSION: This is the first

substantial series to describe the radiological features of paradoxical

neurological TB-IRIS. Compared to published radiological findings of tuberculomas

in HIV-1-infected patients (not receiving ART), an increased inflammatory

response is suggested in tuberculoma IRIS. However, this was not observed in

patients with TB meningitis IRIS.

PMID: 20074410 [PubMed - in process]

 

Validity of symptoms and radiographic features in predicting positive AFB smears

in adolescents with tuberculosis.

Wong KS, Huang YC, Lai SH, Chiu CY, Huang YH, Lin TY.

Department of Paediatrics, Chang Gung Memorial Hospital, Linkou, Taiwan; College

of Medicine, Chang Gung University, Taoyuan, Taiwan. pchest@adm.cgmh.org.tw

SETTING: A cohort of 78 adolescents was selected for evaluation with culture or

histologically proven pulmonary tuberculosis (PTB) from a tertiary paediatric

facility in northern Taiwan. OBJECTIVE: To assess the validity of clinical

features and radiographic findings for predicting positive smears of acid-fast

bacilli (AFB) in adolescents with PTB. DESIGN: A retrospective descriptive study

of adolescents with a confirmed diagnosis of PTB. Clinical symptoms and chest

radiographs were assessed. Univariate analysis identified risk factors suggestive

of a positive AFB smear, and the adjusted odds ratio (aOR) for these features was

calculated using logistic regression. RESULTS: Patients who were AFB

smear-positive and those who were smear-negative differed significantly on

univariate analysis (P < 0.05) with respect to chronic cough, haemoptysis,

multilobar or superior segment of lower lobe involvement, cavitations or presence

of pleural effusions. Logistic regression analysis revealed that risk factors of

positive smear in adolescents with PTB were chronic cough >4 weeks (aOR 13.8,

95%CI 2.3-83.1), lower lobe involvement (aOR 12.6, 95%CI 1.2-134.8) and pulmonary

cavitations (aOR 7.7, 95%CI 1.0-57.7). CONCLUSIONS: For adolescents with PTB,

those suffering from chronic cough for >4 weeks, with involvement of the superior

segment of the lower lobe or with cavitary lesions, have a greater likelihood of

transmitting tuberculosis due to smear positivity.

PMID: 20074405 [PubMed - in process]

 

Diagnosis of drug-resistant tuberculosis: reliability and rapidity of detection.

Van Deun A, Martin A, Palomino JC.

Mycobacteriology Unit, Institute of Tropical Medicine, Antwerp, Belgium;

International Union Against Tuberculosis and Lung Disease, Paris, France.

AVanDeun@theunion.org

With the emergence of multidrug-resistant tuberculosis (MDR-TB), the need for

rapid drug susceptibility testing (DST) is felt globally. National tuberculosis

control programmes (NTPs) may find it hard to choose from the bewildering variety

of rapid tests. We give an overview of the most important methods, discussing

their merits and shortcomings, emphasising techniques that offer an alternative

to the commercial systems and genotypic tests. Correlation between phenotypic and

genotypic DST remains problematic due to our insufficient knowledge of the

mutations underlying drug resistance, besides the past standardisation of

phenotypic DST. Rapid growth-based DST tends to be less accurate due to growth

retardation of some resistant strains. To arrive at optimal resistance monitoring

and management of MDR-TB without overloading the laboratories, the test

indications and definition of a suspect need careful consideration, while

excellent microscopy remains crucial but challenging. The hitherto little-studied

fluorescein diacetate vital staining technique may offer the solution,

reconciling earlier detection and appropriate pre-selection for rapid DST. For

the choice of methods, appropriateness and sustainability should be considered in

conjunction with the prospects for complete population coverage. Excellent

coverage will only be feasible through decentralisation of simple,

low-requirement methods or alternatively by centralised genotypic DST with, in

principle, easy specimen referral. The small differences in DST turnover time are

relatively unimportant, provided primary culture isolation is not required. No

test is fully accurate, and proper pre-selection may allow the use of less

accurate but simple screening methods. Conventional slow DST will still be needed

for confirmation and for epidemiological monitoring.

PMID: 20074402 [PubMed - in process]

 

Evidence for an effect of fetal growth on the risk of tuberculosis.

Villamor E, Iliadou A, Cnattingius S.

Department of Environmental Health Sciences, University of Michigan School of

Public Health, Ann Arbor, Michigan, USA. villamor@umich.edu

We examined the risk of tuberculosis in relation to birth weight and ponderal

index among 21,596 Swedish twins born from 1926 through 1958. Using a cohort

design, tuberculosis risk was 11% lower for every 500 g of birth weight (P = .05)

and 8% lower for every 0.2 of ponderal index, calculated as birth weight in grams

multiplied by 100 and divided by the cube of birth length in centimeters (P =

.08). The association between birth weight and tuberculosis was stronger in male

individuals than in female individuals. In co-twin control analyses among

disease-discordant monozygotic twins, tuberculosis risk was 46% lower for every

500 g of birth weight (P = .05). The association was stronger in male individuals

(87% risk reduction; P = .02) than it was in female individuals (16% reduction; P

= .62). A similarly stronger relation with male sex, compared with female sex,

was found for ponderal index. Because associations among monozygotic twins are

largely independent of shared genetic or environmental factors, we postulate that

fetal growth may play a causal role in susceptibility to tuberculosis, possibly

through early programming of immunity.

PMID: 20047499 [PubMed - indexed for MEDLINE]

 

Limited sampling strategies for therapeutic drug monitoring of linezolid in

patients with multidrug-resistant tuberculosis.

Alffenaar JW, Kosterink JG, van Altena R, van der Werf TS, Uges DR, Proost JH.

Department of Hospital and Clinical Pharmacy, University Medical Center

Groningen, University of Groningen, Groningen, The Netherlands.

j.w.c.alffenaar@apoth.umcg.nl

INTRODUCTION: Linezolid is a potential drug for the treatment of

multidrug-resistant tuberculosis but its use is limited because of severe adverse

effects such as anemia, thrombocytopenia, and peripheral neuropathy. This study

aimed to develop a model for the prediction of linezolid area under the plasma

concentration-time curve from 0 to 12 hours (AUC0-12h) by limited sampling

strategy to enable individualized dosing. PATIENTS AND METHODS: Fourteen patients

with multidrug-resistant tuberculosis received linezolid twice daily as part of

their antituberculosis treatment. Linezolid concentrations were determined at

steady state by high-performance liquid chromatography tandem mass spectrometry

before and at 1, 2, 4, 8, and 12 hours after dosing. Linezolid AUC0-12h

population model and limited sampling models were calculated with MWPharm

software. The correlation between predicted linezolid AUC0-12h and observed

linezolid AUC0-12h was investigated by Bland-Altman analysis. RESULTS: A total of

26 pharmacokinetic profiles were obtained. The median AUC0-12h was 51.8

(interquartile range, 41.8-65.9) mg*h/L at 300 mg and 123.8 (interquartile range,

100.9-152.5) mg*h/L at 600 mg, both twice daily. The most relevant model

clinically for prediction of linezolid AUC0-12h used a linezolid trough

concentration (r = 0.91, prediction bias = -2.9% and root mean square error =

15%). DISCUSSION: The difference between choosing a trough concentration and two

to three samples increased the correlation from 0.90 to 0.95 but appeared not

clinically relevant because it did not result in different dosing advice.

CONCLUSION: This study showed that linezolid AUC0-12h in patients with

multidrug-resistant tuberculosis could be predicted accurately by a minimal

sampling strategy and could be used to individualize the dose.

PMID: 20042919 [PubMed - in process]

 

To catch a killer. What can mycobacterial models teach us about Mycobacterium

tuberculosis pathogenesis?

Shiloh MU, DiGiuseppe Champion PA.

Department of Medicine, Division of Infectious Diseases, University of

California, San Francisco, CA 94158, USA.

Mycobacterium tuberculosis is the causative agent of the global tuberculosis

epidemic. To combat this successful human pathogen we need a better understanding

of the basic biology of mycobacterial pathogenesis. The use of mycobacterial

model systems has the potential to greatly facilitate our understanding of how M.

tuberculosis causes disease. Recently, studies using mycobacterial models,

including M. bovis BCG, M. marinum, and M. smegmatis have significantly

contributed to understanding M. tuberculosis. Specifically, there have been

advances in genetic manipulation of M. tuberculosis using inducible promoters and

recombineering that alleviate technical limitations in working with mycobacteria.

Model systems have helped elucidate how secretion systems function at both the

molecular level and during virulence. Mycobacterial models have also led to

interesting hypotheses about how M. tuberculosis mediates latent infection and

host response. While there is utility in using model systems to understand

tuberculosis, each of these models represent distinct mycobacterial species with

unique environmental adaptations. Directly comparing findings in model

mycobacteria to those in M. tuberculosis will illuminate the similarities and

differences between these species and increase our understanding of why M.

tuberculosis is such a potent human pathogen. Copyright 2009 Elsevier Ltd. All

rights reserved.

PMID: 20036184 [PubMed - in process]

 

PLGA microparticles in respirable sizes enhance an in vitro T cell response to

recombinant Mycobacterium tuberculosis antigen TB10.4-Ag85B.

Shi S, Hickey AJ.

Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North

Carolina at Chapel Hill, Chapel Hill, North Carolina 27599-7571, USA.

PURPOSE: To study the use of poly (lactide-co-glycolide) (PLGA) microparticles in

respirable sizes as carriers for recombinant tuberculosis (TB) antigen,

TB10.4-Ag85B, with the ultimate goal of pulmonary delivery as vaccine for the

prevention of TB. MATERIALS AND METHODS: Recombinant TB antigens were purified

from E. coli by FPLC and encapsulated into PLGA microparticles by

emulsion/spray-drying. Spray-drying condition was optimized by half-factorial

design. Microparticles encapsulating TB antigens were assessed for their ability

to deliver antigens to macrophages for subsequent presentation by employing an in

vitro antigen presentation assay specific to an Ag85B epitope. RESULTS:

Spray-drying condition was optimized to prepare PLGA microparticles suitable for

pulmonary delivery (aerodynamic diameter of 3.3 microm). Antigen release from

particles exhibited an initial burst release followed by sustained release up to

10 days. Antigens encapsulated into PLGA microparticles induced much stronger

interleukin-2 secretion in a T-lymphocyte assay compared to antigen solutions for

three particle formulations. Macrophages pulsed with PLGA-MDP-TB10.4-Ag85B

demonstrated extended epitope presentation. CONCLUSION: PLGA microparticles in

respirable sizes were effective in delivering recombinant TB10.4-Ag85B in an

immunologically relevant manner to macrophages. These results set the foundation

for further investigation into the potential use of PLGA particles for pulmonary

delivery of vaccines to prevent Mycobacterium tuberculosis infection.

PMID: 20024670 [PubMed - in process]

 

Expression and molecular characterization of the Mycobacterium tuberculosis PII

protein.

Bandyopadhyay A, Arora A, Jain S, Laskar A, Mandal C, Ivanisenko VA, Fomin ES,

Functional Genomics Unit, Institute of Genomics and Integrative Biology (CSIR),

Delhi 110 007, India.

The signal transduction protein PII plays an important role in cellular nitrogen

assimilation and regulation. The molecular characteristics of the Mycobacterium

tuberculosis PII (Mtb PII) were investigated using biophysical experiments. The

Mtb PII coding ORF Rv2919c was cloned and expressed in Escherichia coli. The

binding characteristics of the purified protein with ATP and ADP were

investigated using surface plasmon resonance (SPR) and isothermal titration

calorimetry (ITC). Mtb PII binds to ATP strongly with K(d) in the range 1.93-6.44

microM. This binding strength was not significantly affected by the presence of

2-ketoglutarate even in molar concentrations of 66 (ITC) or 636 (SPR) fold excess

of protein concentration. However, an additional enthalpy of 0.3 kcal/mol was

released in presence of 2-ketoglutarate. Binding of Mtb PII to ADP was weaker by

an order of magnitude. Binding of ATP and 2-ketoglutarate were analysed by

docking studies on the Mtb PII crystal structure (PDB id 3BZQ). We observed that

hydrogen bonds involving the gamma-phosphate of ATP contribute to enhanced

binding of ATP compared with ADP. Glutaraldehyde crosslinking showed that Mtb PII

exists in homotrimeric state which is consistent with other PII proteins.

Phylogenetic analysis showed that Mtb PII consistently grouped with other

actinobacterial PII proteins.

PMID: 19884192 [PubMed - in process]

 

Rational design of multiple TB antigens TB10.4 and TB10.4-Ag85B as subunit

vaccine candidates against Mycobacterium tuberculosis.

Shi S, Yu L, Sun D, Liu J, Hickey AJ.

Molecular Pharmaceutics, School of Pharmacy, University of North Carolina at

Chapel Hill, Chapel Hill, North Carolina 27599-7360, USA.

PURPOSE: Rational design of recombinant antigens TB10.4 and TB10.4-Ag85B as

subunit vaccine candidates against Mycobacterium tuberculosis. The main purpose

is to obtain a large quantity of soluble antigens. METHODS: Recombinant antigens

were cloned in frame with the N-terminal thioredoxin and expressed in E. coli.

The thioredoxin tag was removed by TEV protease. Nickel-affinity and

size-exclusion chromatography were used to purify antigens to homogeneity.

Antigen stability at different pH levels was studied by photon correlation

spectrometry. Circular dichroism was used to probe antigen secondary structure

and thermal stability. RESULTS: N-terminal thioredoxin fusion dramatically

increased antigen solubility. Soluble TB10.4 and TB10.4-Ag85B were purified to

homogeneity and obtained in milligram quantity. Co-expression of bacteria

chaperons increased the yield of TB10.4-Ag85B. Soluble TB10.4 and TB10.4-Ag85B

purified from the inclusion body showed a reversible structure change. However,

Ag85B and soluble TB10.4-Ag85B showed a clear melting temperature, above which

the secondary structure was lost dramatically. CONCLUSION: Soluble TB10.4 and

TB10.4-Ag85B were purified from the E. coli in significant quantities. The

methods to purify and characterize these recombinant antigens were established,

which paved the way for further vaccine development based on these antigens.

PMID: 19862606 [PubMed - in process]

 

The quest for biomarkers in tuberculosis.

Parida SK, Kaufmann SH.

Max Planck Institute for Infection Biology, Department of Immunology,

Charitéplatz 1, D-10117 Berlin, Germany. parida@mpiib-berlin.mpg.de

No new vaccine has been licensed for tuberculosis (TB) for more than

three-quarters of a century, and no new drug has been licensed for half a

century. One major drawback has been the attrition caused by the lack of a

reliable biological indicator (biomarker) to predict toxicity and efficacy early

in the development pipeline. This review portrays the landscape of biomarker

discovery for TB in the context of drug and vaccine development using emerging

global biomics platforms. The time is ripe to move from single markers for

correlates of protection to a biosignature comprising a well-defined set of

robust indicators in TB that can accelerate rapid screening and early selection

of potential drug and vaccine candidates. Copyright (c) 2009 Elsevier Ltd. All

rights reserved.

PMID: 19854295 [PubMed - in process]

 

Utility of a combination of RD1 and RD2 antigens as a diagnostic marker for

tuberculosis.

Kalra M, Khuller GK, Grover A, Behera D, Wanchu A, Verma I.

Department of Biochemistry, Postgraduate Institute of Medical Education and

Research, Chandigarh, India.

We evaluated the diagnostic potential of a cocktail of 4 antigens encoded by

regions of difference (RD) 1 and 2 of Mycobacterium tuberculosis, that is, early

secretory antigenic target-6, culture filtrate protein-10 (CFP-10), CFP-21, and

mycobacterial protein from species tuberculosis-64 (MPT-64) on the basis of

antigen and antibody detection by enzyme-linked immunosorbent assay. Parallel

detection of antigens and antibodies in the serum samples of pulmonary

tuberculosis (PTB) patients resulted in higher sensitivity as compared to either

of the single tests in both smear-positive (90%) and smear-negative (60%) PTB

patients. In addition, combined detection of antigens and antibodies in the

fluids of extrapulmonary tuberculosis (EPTB) patients could detect >90% of the

patients with high specificity. These results demonstrate the ability of the

combination of antigen and antibody detection assays based on the cocktail of RD

antigens to diagnose a substantial number of PTB and EPTB cases with high

specificity. 2010 Elsevier Inc. All rights reserved.

PMID: 19833469 [PubMed - in process]

 

Past, present and future directions in human genetic susceptibility to

tuberculosis.

Möller M, de Wit E, Hoal EG.

Molecular Biology and Human Genetics, MRC Centre for Molecular and Cellular

Biology and the DST/NRF Centre for Biomedical TB Research, Faculty of Health

Sciences, Stellenbosch University, Tygerberg, South Africa.

The historical impression that tuberculosis was an inherited disorder has come

full circle and substantial evidence now exists of the human genetic contribution

to susceptibility to tuberculosis. This evidence has come from several

whole-genome linkage scans, and numerous case-control association studies where

the candidate genes were derived from the genome screens, animal models and

hypotheses pertaining to the disease pathways. Although many of the associated

genes have not been validated in all studies, the list of those that have been is

growing, and includes NRAMP1, IFNG, NOS2A, MBL, VDR and some TLR. Certain of

these genes have consistently been associated with tuberculosis in diverse

populations. The future investigation of susceptibility to tuberculosis is almost

certain to include genome-wide association studies, admixture mapping and the

search for rare variants and epigenetic mechanisms. The genetic identification of

more vulnerable individuals is expected to inform personalized treatment and

perhaps vaccination strategies.

PMID: 19780822 [PubMed - in process]

 

Clinical efficacy of direct DNA sequencing analysis on sputum specimens for early

detection of drug-resistant Mycobacterium tuberculosis in a clinical setting.

Choi JH, Lee KW, Kang HR, Hwang YI, Jang S, Kim DG, Kim CH, Hyun IG, Shin TR,

Division of Pulmonary, Allergy and Critical Care Medicine, Department of Internal

Medicine, Hallym University Medical Center, Gangwon-do, Korea.

BACKGROUND: Early detection of drug-resistant Mycobacterium tuberculosis is

important for the control and prevention of disease transmission. However,

conventional drug susceptibility tests for drug-resistant M tuberculosis take at

least 3 to 8 weeks. Here, we report the clinical efficacy of direct DNA

sequencing analysis for detecting drug-resistant TB on sputum specimens in a

clinical setting. METHODS: A total of 113 sputum specimens from 111 patients, who

were suspected of having drug-resistant TB by clinicians, were used for DNA

sequencing of katG, rpoB, embB, and pncA genes for isoniazid (INH), rifampin

(RIF), ethambutol (EMB), and pyrazinamide (PZA) resistance, respectively, and the

results were compared with drug susceptibility tests. The optimization of

antituberculosis drugs according to the results of DNA sequencing and the

treatment outcomes of the patients were also analyzed. RESULTS: Turnaround time

of the direct DNA sequencing analysis was 3.8 +/- 1.8 days. We found mutations

related to drug resistance in 30 clinical specimens for katG, 39 for rpoB, 13 for

embB, and 24 for pncA. The sensitivity and specificity of the assay were 63.6%

and 94.6% for INH, 96.2 and 93.9% for RIF, 69.2% and 97.5% for EMB, and 100% and

92.6% for PZA, respectively. Of the patients with RIF resistance, including

multidrug-resistant TB by the assay, 92.5% of the patients with initial

first-line antituberculosis drugs were changed to second-line antituberculosis

drugs, and treatment was successful in 61.9% of these cases. CONCLUSION: Direct

DNA sequencing analysis of clinical sputum specimens is a rapid and useful method

for the detection and treatment of drug-resistant TB.

PMID: 19741059 [PubMed - indexed for MEDLINE]